Alzheimer?s Disease (AD), the most common cause of dementia, increases with age, afflicts 5.5 million people in the US, and there is no cure. This phase I project will initiate development of the Alzheimer?s Therapeutics Screening Assay (ATSA), which will utilize neurons, microglia, and astrocytes, derived from human induced pluripotent stem cells. A new testing system is desperately needed as previously FDA-approved therapeutics for AD have limited and temporary effects, and no new drug has been approved for AD since 2003; the many failed clinical trials since then illustrate that current preclinical models are not sufficiently predictive for AD. As amyloid beta peptide (A?) plaques accumulate in the brains of AD patients, pharmaceutical companies have exhaustively targeted the beta-amyloid cascade pathway for drug-discovery. However, while many of these agents reduced plaques, none effectively slowed or prevented memory loss in clinical trials. Functional magnetic resonance (fMRI) studies show that subjects with amnestic mild cognitive impairment (aMCI), a prodromal symptom of AD, display hyperactive hippocampal neurons prior to plaque formation, suggesting that dysregulation of neuronal calcium may underlie AD. Relatedly, microglia, the resident immune cells of the brain, are activated in AD and secrete glutamate and inflammatory cytokines that may alter neuronal calcium. Furthermore, the greatest genetic risk for AD is isoform 4 of apolipoprotein E (APOE4), and proteolytic fragments of APOE4 cause dysregulation of intracellular calcium and are toxic to neurons and microglia. For the ATSA, methods will be developed in which hiPSC-neurons, -MG, and -astrocytes are cocultured and AD- relevant stresses (e.g., APOE4 fragments or A? oligomers) added, and the cells monitored for alterations in calcium (or voltage) using Kinetic Image Cytometry, a digital microscopy method that quantifies calcium transient activity on a cell-by-cell basis. Cell morphology (neurites, synaptic puncta) and relevant biomarkers will also be quantified. The ATSA will be versatile, and phase II goals will include utilizing iPSCs from patients with inherited or sporadic AD, representing both genders, will be incorporated to further interrogate AD-relevant pathways. Once developed, the ATSA platform will enable testing of hundreds of compounds per day to identify those with beneficial effects against AD related stresses. The ATSA will be marketed by Vala Sciences Inc for contract research to pharmaceutical companies developing novel therapeutics for AD, and used for internal drug-discovery by Vala researchers.